The invention relates to enzyme immunoassays, and more particularly to self-contained, one-step assays.
Immunoassays for the detection of antigens are used extensively throughout the world in clinical and medical applications. Common uses include indication of pregnancy and ovulation, and infectious disease. Historically, these assays have been performed by skilled laboratory technicians, entailing the use of numerous reagents in a complicated sequence of steps. Recently, great efforts have been directed towards developing assays which are simpler, less expensive, and more reliable. For example, assay devices now exist which can be used by non-skilled individuals, in non-laboratory environments. These devices, however, are greatly in need of improvement. Problems include complicated steps, difficult interpretation, limited storage life, and unreliability.
One such assay is disclosed in U.S. Pat. No. 4,446,232 to Liotta. The assay therein disclosed has three layers: a first containing an enzyme labelled antibody specific to the test antigen; a second containing bound purified antigen of the type tested for; and a third containing a substrate which reacts with an enzyme bonded to the antibody. This assay suffers from the disadvantage of requiring high antigen purity. Most commonly used antigens, for example HCG, are quite costly to obtain and purify in high quantity. The result is a high cost of manufacture, and a correspondingly high cost to the consumer.
An additional disadvantage of the '232 assay is that both monovalent binding sites on the antibody must be antigen linked for a positive reading. Accordingly, accurate readings require high antigen concentrations, whereby the assay has a low sensitivity.
Moreover, all antigen-linked antibody, and correspondingly all enzyme, diffuses throughout a substrate region, whereby a faint color reaction results. Thus, results are difficult to interpret, and do not provide quantitative information.
It is therefore an object of the invention to provide a single-step enzyme linked immunoassay device which can be easily carried out by a non-skilled user.
It is a further object to provide for higher sensitivity than heretofore known.
It is an additional object to provide for reduced device cost, by employing easily purified, low cost components.
It is yet another object of the invention to provide for sharp, easily readable color indication, while rendering clear quantitative information.